Enterovirus A71 capsid protein VP1 increases blood–brain barrier permeability and virus receptor vimentin on the brain endothelial cells

Figures

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  Fig. 1. VP1 increased BBB permeability in vivo. a Western blotting showed the increased albumin in the brain tissues from the mice treated with VP1. b Immunohistochemistry staining revealed evident albumin in the brain tissues from the mice treated with VP1. c, d Fluorescence intensity of Evans blue dye (EBD) or FD4 was significantly elevated in the brain tissues from the mice treated with VP1. e Expression levels of tight junction proteins in the brain tissues. ZO-1 was comparable in the mice treated with normal saline or VP1. Claudin-5 was significantly reduced in the brain tissues treated with VP1. *p < 0.05

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  Fig. 2. VP1 reduced claudin-5 and disrupted BBB in vitro. a VP1 significantly decreased the trans-endothelial electrical resistance (TEER). b VP1 damaged the tight junctions between endothelia cells observed with transmission electron microscope. c, d VP1 directly decreased claudin-5 in the brain endothelial cells; in contrast, ZO-1 was comparable in different groups. **p < 0.01

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  Fig. 3. VP1 promoted the expression of vimentin in the brain tissues. a qRT-PCR showed the vimentin mRNA was significantly increased in the brain tissues from the mice treated with VP1. b Western blotting further showed the vimentin protein was significantly increased in the brain tissues from the mice treated with VP1. c In the immunohistochemistry analysis, vimentin was widely distributed in brain tissues and significantly increased upon VP1 challenge. **p < 0.01. d EV-A71 receptors SCARB2 and HSP-70 were almost unchanged on the brain endothelia cells treated with VP1. e EV-A71 receptor Annexin II was slightly decreased on the brain endothelia cells treated with VP1. Expression of PSGL-1 was comparable in the different groups

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  Fig. 4. VP1 increased the expression of vimentin on the brain endothelial cells. a A dose-dependent increase of vimentin on the endothelial cells treated with VP1 for 24 h. VP1 with 0.1 μg/ml was enough to stimulate the expression of vimentin. b A time-dependent increase of vimentin on the endothelial cells treated with 0.1 μg/ml VP1. c Immunofluorescence analysis showed the enhanced expression of vimentin on endothelial cells treated with VP1 for 24 h. d VP1 neutralization antibody competitively inhibited the roles of VP1 in the upregulation of vimentin. Ctrl-Ab control antibody, Anti-VP1 VP1 neutralization antibody. e, f Expression of EV-A71 receptors SCARB2, HSP-70, and Annexin II was similar in the different groups. PSGL-1 was decreased in the endothelia cells treated with VP1

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  Fig. 5. TGF-/Smad3 and NF-κB-dependent expression of vimentin on brain endothelial cells treated with VP1. a In the VP1-treated endothelial cells, TGF-β and Smad-3 was increased and activated. b NF-κB p65 was activated; and STAT3 was not changed. c NF-κB inhibitor BAY or Smad-3 inhibitor SIS3 suppressed the elevation of vimentin on the VP1-treated endothelial cells

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  Fig. 6. Vimentin over-expression damaged BBB integrity. a Vimentin expression plasmid promoted the expression of vimentin on brain endothelial cells. b Vimentin expression plasmid transfection increased the FD4 leakage through brain endothelial cell monolayer. ***p < 0.001